EFFECT of the KANAMYCIN RESISTANCE MARKER on STABILITY of 2M-BASED EXPRESSION PLASMIDS

Arch. Biol. Sci., Belgrade, 59 (1), 1-12, 2007. DOI:10.2298/ABS0701001S

EFFECT OF THE KANAMYCIN RESISTANCE MARKER ON STABILITY OF 2m-BASED EXPRESSION PLASMIDS

STANKOVIĆ NADA., VASILJEVIĆ BRANKA., and†G. LJUBIJANKIĆ

Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia

Abstract – In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid mainte- nance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS – driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene.

Key words: 2μm plasmid, kanamycin resistance, penicillin G amidase, plasmid stability, Saccharomyces cerevisiae, selective markers.

UDC577.182.76 : 577.21 579.25

INTRODUCTION based expression vectors were successfully applied in our laboratory for expression of the recombinant penicillin G Expression vectors based on the naturally occurring 2μm amidase (penicillin acylase, PGA, pac) gene from the plasmid are the sole vectors in baker’s yeast which satis- bacterium Providencia rettgeri (Ljubijankićet al., fy two major biotechnological requirements: mitotic sta- 1999; Ljubijankićet al., 2002) and gene for human bility and high copy number. Vectors that employ the interferon b (Todorović et al., 2000) in baker’s whole 2μm sequence have proved to be the most stable yeast. YEps (yeast episomal plasmid), since they contain all the sequences that control plasmid DNA replication and par- During our work with various Yeps, we realized the titioning. However, artificially constructed 2μm-based necessity for quick and exact monitoring of the mitotic vectors appear to be less stable than native 2μm plasmids, stability of plasmids. For vectors whose presence is diffi- and are maintained at a lower copy number (Futcher cult or expensive to detect via a recombinant product, the and Cox, 1984). Alternative methods in yeast expres- introduction of a dominant selective marker is a logical sion vector design include disintegrative vectors, which alternative. Despite much effort to introduce novel mark- are recombination cointegrants made up of a 2μm-based er genes (as listed in van den Berg and vector and pRL (Chinerry and Hinchliffe, Steensma, 1997) into yeast biotechnology, there are 1989). In the yeast cell, the cointegrant is resolved by few real dominant selective markers in S. cerevisiae, and (recombinase)-mediated FRT recombination into its two their application is restricted. Although the majority of components. An Escherichia coli plasmid carrying the selective markers cannot be applied for large-scale pro- yeast LEU2 marker and a single FRT (FLP recognition duction, their role in construction and selection of the target) sequence (Bruschi and Howe, 1988), pRL is optimal productive clones is irreplaceable. In order to subsequently lost from the population because it is retain a high yield of biomass, we decided to introduce unable to propagate in S. cerevisiae. Disintegrative 2μm- the dominant selective marker gene for aminoglycoside

1 2 N. STANKOVIĆ ETAL. phosphotransferase I (APT I, Kmr), which confers resist- BLITZ cassette is driven by the strong and strictly regu- ance to kanamycin in prokaryotes and G418 in eukary- lated GAL1-10 promoter, which is repressed in the pres- otes (Jimenez and Davies, 1980). The ence of glucose and induced about 1000-fold by galac- KanMX4 cassette (Wach et al., 1996) that we used has tose (Romanos et al., 1992). a dual (bacterial/fungal) promoter. The strong constitutive TEF promoter from the fungus Ashbya gossypii The aim of this study was to test the dominant marker gene for G418 resistance as a useful tool for monitor- allows effective expression of the Kmr gene in yeast. A ing the proportion of plasmid-bearing cells in culture, single copy of Kmr in A. gossypii confers resistance for while simultaneously examining the influence of the up to 8 mg/ml of geneticin (Steiner and selective marker on YEp stability. Philippsen, 1994).

However introducing a new gene could burden plas- MATERIALS AND METHODS mid expression and plasmid mitotic stability, consequent- ly decreasing the yield of the recombinant product. In Bacterial and yeast strains order to examine the effect(s) of the dominant selective – marker Kmr on vector stability, various E.coli/S. cerevisi- The E. coli strain DH5a (F Dlac U169 (F80 lacZ ae shuttle vectors were constructed based on disintegra- DM15) supE44 hsdR17 recA1 gyrA96 endA1 thi-1 relA1) tive plasmids pBLU-D (Ludwig and Bruschi, (Hanahan 1983) was used to clone all the plasmids and 1991) and pGoB-2 (Ljubijankić et al., 1999). constructs described in this work. The S. cerevisiae strain Plasmid pBLUR-D, with the yeast auxotrophic URA3 CBL1-30 (MATα [cirº] pep4-3 his3Δ::GAL10p-GAL4- marker gene, is the resolved form of pBLU-D (Fig. 1) URA3 leu2-3,112 trp1-289 ura3-52 canR), harboring and appears to be maintained at a high copy number with- plasmids pGoBR-2KS and pGIFNR (Todorović et al., out selective pressure. Construct pGoB-2 (Fig. 2) is the 2000), has been described previously (Ludwig 1991). construct for pac expression in which maximal recombi- Isogenic ura– strain GSP-3 (MATα [cirº] pep4-3 nant enzyme yield is provided by the BLITZ expression his3Δ::GAL10p-GAL4-ura3Δ247 leu2-3,112 trp1-289 cassette (Ludwig et al., 1993). Expression from the ura3-52 canR) was constructed as described previously

r Fig. 1 - pBLU-based disintegrative vectors. The Km gene was cloned in the SmaI site of pBLU-D with or without different TRPter sequences in different orientations. The ure of pBLU-D was provided by courtesy of C. V. Bruschi. EFFECT OF KANAMYCINE RESISTANCE MARKER 3

Fig. 2 - 2mm based vectors with the BLITZ expression cassette: a- Vector pBLAST gained after removing the lacZ reporter gene from pBLITZ contains the “empty” BLITZ expression cassette with SmaI and BamHI sites for foreign gene insertion (Ludwig et al., 1993). b - Different sequences inserted into the expression cassette resulted in different expression vectors. The 3’f pa - pac gene was cloned together with the downstream fragment of the P. rettgeri chromosome. The figure of pBLAST was provided by courtesy of C. V. Bruschi.

(Pavković & Ljubijankić, 2000) in order to Media and culture conditions provide an appropriate genetic background for testing the plasmid stability of URA3 carrying pBLU-based vectors. Esherichia coli strains were grown in LB medium Briefly, the URA3 gene on the CBL1-30 chromosome with or without agar (20 g/L). Ampicillin was added at was replaced with the inactive allele ura3D247, which 100 µg/µl and kanamycin at 60 µg/µl for selection of was obtained by deletion of the 247 bp StuI/EcoRV frag- recombinant plasmids. Saccharomyces cerevisiae strains ment of the URA3 gene on the YIplac211 plasmid were grown in one of the following standard yeast media: (Gietz and Sugino, 1988). The inactive allele semidefined rich YPD (1% yeast extract, 2% peptone, was introduced into competent CBL1-30 yeast cells by 2% glucose), YPRaf (1% yeast extract, 2% peptone, 2% electroporation. The Ura– transformants were scored on raffinose), and YPGal (1% yeast extract, 2% peptone, 2% galactose), with or without addition of 200 µg/µl of G418 DOura– medium with 5-fluoroorotic acid (Boeke et (geneticin, G418 sulfate Gibco BRL, Gaithersburg, MD); al., 1984). 4 N. STANKOVIĆ ETAL. and the defined yeast synthetic complete media, leucine- described by Inoueet al. (1990). Yeast transformation less (DOleu–) and uracil-less (DOura–), described by by electroporation was performed according to Sherman et al. (1986). For pac expression, a self- Meilhocet al. (1990). The transformation efficiency inductive medium was used (YPGal with addition of of S. cerevisiae was in the order of magnitude of 103 0.2% glucose). transformants/mg of DNA. Escherichi coli plasmid DNA was isolated using the QIAGEN Plasmid Kit and the DNA transformation and recombinant DNA techniques QIAGEN Mini-prep Kit (QIAGEN) or as described by Del Sal et al. (1989). Manipulations of DNA such as The DH5α E. coli strain was transformed as restriction enzyme digestions, ligations and agarose gel

Table 1. List of plasmids used in this work. a See text for details.

E. coli vectors

Plasmid Relevant characteristica Source or reference:

pFAKanMX4 Apr, Kmr Wach et al., 1996

pUC18/19 Apr Yanisch-Perron et al., 1985 r pUC19T Ap , S. c. TRP1ter Storici, F.

r r pUC18TK Ap , S. c. TRP1ter, Km this paper

r r pUC19TK Ap , S. c. TRP1ter, Km this paper

r r pUC18KT5 Ap , S. c. TRP5ter, Km this paper

r r pUC19KT5 Ap , S. c. TRP5ter, Km this paper

E. coli / S. cerevisiae shuttle vectors

Plasmid Relevant characteristica Source or reference:

pBLU-D pRL + URA3 Ludwig and Bruschi, 1991

pBLU-KP pRL + URA3, Kmr this paper

pBLU-KS pRL + URA3, Kmr this paper

r pBLU-KT5P pRL + URA3, Km , TRP5ter this paper

r pBLU-KT5S pRL + URA3, Km , TRP5ter this paper

r pBLU-TK pRL + URA3, Km , TRP1ter this paper

r r YCpKan pRL + Ap , Km , BLITZ cassette, TRP5ter Todorović et al., 2000

pGIFN pRL + Kmr, BLITZ cassette, hIFN-b Todorović et al., 2000

pGoB-2 pRL + BLITZ cassette, P. r. pac Ljubijankićet al., 1999

pGoB-2KS pRL + Kmr, BLITZ cassette, P. r. pac Todorović et al., 2000

EFFECT OF KANAMYCINE RESISTANCE MARKER 5 electrophoresis were performed as described by r 2681 bp BglII/SalI Km TRP5ter cassette from YCpKan Maniatis et al. (1982). was first inserted into pUC19 digested with BamHI and SalI restriction enzymes and then recovered as a 1827 bp DNA hybridization fragment by digestion with SmaI and ligated in both ori- Total yeast DNA was isolated according to entations to pBLU-D previously linearized with SmaI. Ausubelet al. (1989), digested with EcoRI, trans- Resolution of vectors after transformation ferred from an agarose gel onto a nylon membrane, and hybridized by the Southern procedure (Maniatis et A GSP-3 strain was transformed with pBLU-based al., 1982). Plasmid DNA was detected with a probe com- vectors. Immediately after transformation, the cells were r prising the 428 bp HindIII/NcoI fragment of the Km plated onto DOleu– plates. When colonies appeared, at gene. The probe for genomic DNA was synthesized from least three individual transformants of each plasmid type S. cerevisiae gene VMA22 (generated by a PCR with were picked and cultivated for 30 generations in YPD primers 5’-AGGATCCGGCTTAACGAGAAG-3’ and medium in order to allow full resolution of disintegrative 5’-AGGATCCTTTTTCAAACCATGGAC-3’). plasmids and loss of pRL from the population. Cultures We decided to use the VMA22 gene probe as a con- were then diluted and plated onto YPD plates to yield trol because it is present in one copy per haploid yeast ~200 colonies per plate. Colonies were subseqently test- genome (Skoko et al., 2005) and primers were already ed for the loss of the carrier plasmid pRL by replica-plat- available. The DNA probes were labeled with [α- ing (as mini streaks) onto DOleu– minimal medium, and 32P]dCTP by random primer extension (Multiprime DNA for the presence of resolved expression plasmids labeling system, Amersham). Hybridization was detected pBLUR-KP, pBLUR-KS, pBLUR-TK, pBLUR-KT5P, by autoradiography, and the plasmid copy number was and pBLUR-KT5S by replica plating onto YPDG418 determined densitometrically by comparing the signal of selective plates. the single-copy gene to the plasmid DNA signal in the same lane (BioDocAnalyze 1.0, Biometra, Göttingen, Determination of 2ìm-based plasmid stability Germany). Plasmid stability was determined according to the Plasmid construction modified method of Mann and Davies (Gietz & Sugino, 1988) by cultivating on selective and nonse- A list of plasmids used in this study is given in Table lective media. Testing in non-selective conditions was 1. The plasmid pBLU-D was used for all subsequent con- expanded to a nonselective repressive medium with glu- structions described in this paper. Construction of the cose (YPD) and a nonselective inductive medium with plasmids YCpKan, pGoB-2KS, and pGIFN was galactose (YPGal). Cells were cultivated for 12 genera- described previously (Todorovićet al., 2000). tions in YPRafG418 – a selective medium with a neutral 1. Plasmids pBLU-KP and pBLU-KS: the1483 bp carbon source – to ensure plasmid presence in the popu- EcoRV/SmaIfragment with the KanMX4 cassette from lation and to avoid the effects of glucose and galactose on pFAKanMX4 was inserted in both orientations into the GAL1-10UAS driven expression. A portion of the culture pBLU-D vector digested with SmaI. was diluted and plated onto YPD plates to give 200-300 colonies per plate for monitoring of plasmid presence in r 2. Plasmid pBLU-TK: the Km gene obtained as a the starter culture. Inocula (0.02%) of the starter culture ScaI/SmaI fragment from pFAKanMX4 was ligated with (3x105 cell/ml) were transferred to parallel YPD and the pUC19T vector linearized with SmaI to give YPGal cultures and cultivated for 36 generations. On the r pUC19TK. The Km TRP1ter cassette obtained by diges- basis of our previous work in establishing optimal culture tion of pUC19TK with SphI and NdeI was inserted into conditions for pac expression (Ljubijankić et al., pUC18 digested with the corresponding enzymes, after 2002), we concluded that a 36-generation cultivation is which pUC18TK was then digested with SmaI and the sufficient for plasmid instability. To maintain cultures in r obtained 1624 bp Km TRP1ter cassette was ligated into log phase, 0.02% inocula of the cultures were transferred pBLU-D previously linearized with SmaI. to fresh media (batches I, II, and III) every 12 generations. To monitor plasmid stability, a part of the cultures 3. Plasmids pBLU-KT5P and pBLU-KT5S: the from the first and the third batch were diluted and plated 6 N. STANKOVIĆ ETAL. onto YPD plates to yield 200-300 colonies per plate. and the plasmid stability assay was performed in the Colonies from YPD plates were then replica-plated onto media with optimal (glucose) and slow (galactose) selective YPDG418 plates and the number of colonies growth rates for all vectors with or without GALUAS. grown on the YPD plates (100%) was compared to the number of colonies grown on the selective plates. Resolution of vectors after transformation Plasmid stability was calculated from the formula given In the case of pBLU-based plasmids, after thirty by Christiansonet al. (1992): generations of nonselective growth less than 20% of the + + ln(P2/P1) cells contained both selective markers (Leu G418 phe- PLR = 1- e g [plasmid/(cell)(division)] notype), while the remaining cells contained only the Kmr marker (Leu–G418+ phenotype). Colonies with the PLR is the plasmid loss rate; P1 is the number of Leu–G418+ phenotype were chosen to determine the sta- colonies grown on the nonselective plate; P2 is the num- bility of 2μm-based plasmids alone. For this reason from ber of colonies grown on the selective plate; and g is the this point the letter “R” is added to plasmid names refer- number of generations. ring to the resolved plasmid form.

The determined plasmid loss rate expressed as the The stability of pGoB-based vectors number of plasmid-free segregants in the population after a single division is the mean of at least two independent Stability of pGoBR-2 was estimated during rPGA experiments. Cultures were subsequently pelleted and production (Ljubijankić et al. 2002), and the num- total yeast DNA was isolated for estimation of the plas- ber of plasmid-bearing cells was estimated via the num- + mid copy number. ber of PGA clones. Comparison of the stabilities between the parental pGoBR-2 and plasmid pGoBR-2KS pac expression and assay of PGA activity showed that in noninductive conditions pGoBR-2 was completely stable (99% of PGA+ clones), while pGoBR- A mesured volume (10 ml) of self-inductive medium 2KS had a plasmid loss rate of 5.15% per generation. was inoculated from the first overnight YPRaf cul- G418 Estimated plasmid loss for pGoBR-2 was 15% per gener- tures of pac carrying clones (pGoBR-2/CBL1-30, ation in the first 24 h after galactose induction (Ljubi- pGoBR-2KS/CBL1-30). After 84 h of growth, 1 ml was jankić, unpublished results). As for pGoBR-2KS, it subtracted to measure rPGA activity using a standard col- orimetric method (Alkema et al., 1999). One unit of enzyme activity was defined as the quantity of PGA cat- Table 2. Loss rates of pGoB-based expression vectors expressed as % of plasmid free cells in population after one division. alyzing hydrolysis of 1 μmol of 6-nitro-3(phenylacetami- PLR - plasmid loss rate, calculated as described in Materials and do) benzoic acid per minute at 25ºC. Methods.

plasmid media PLR/generation (%) RESULTS YPD 5.15±0.41 Growth rates pGoBR-2KS YPGal 14.02 Beyond the inducing effect on GAL promoters, galactose also has a general effect on the growth rate of YPD 2.46±0.32 yeast cultures. Growth rates for CBL1-30 and GSP-3 strains in rich media with different carbon sources (2% pGIFNR YPGal 7.60±2.40 raffinose, 2% glucose, and 2% galactose) were measured (data not shown). We confirmed that the isogenic strains CBL1-30 and GSP-3 had the same generation time, approximately 2 h in glucose and raffinose, and approxi- showed a similar loss rate of approximately 15% per gen- mately 8 h in galactose (data not shown). Plasmid stabil- eration when cultivated in galactose, white pGIFNR ity experiments were therefore performed in the same exhibited plasmid loss rates of 2.46% on glucose and conditions regarding the carbon source: the starter culture 7.60% on galactose and appeared to be twice as stable as had an optimal growth rate in a neutral carbon source, isogenic pGoBR-2KS (Table 2). EFFECT OF KANAMYCINE RESISTANCE MARKER 7

Comparison of rPGA production ture growth conditions and constant selective pressure, the production of rPGA by the pGoBR-2KS clone was Clones carrying vectors with the BLITZ expression barely one third of the production by the clone carrying cassette for the production of recombinant PGA were cul- pGoBR-2. The low activity of the pGoBR-2KS clone in tivated and induced for foreign protein production as producing rPGA is probably due to its diminished stabil- described in Materials and Methods. In self-inductive ity in glucose, resulting in a small number of cells in the medium after 12 h of cultivation, yeast cells exhaust all population containing the pac gene at the beginning of the glucose and switch to utilization of galactose, leading induction. Additionally, the production of rPGA per cell to pac induction. Activities of PGA after 72 h of induc- did not differ considerably between pGoBR-2KS and tion are shown in Fig. 3a. Even after optimization of cul- pGoBR-2 clones (Fig. 3b).

a Stability of pBLU-based vectors

To define the direct contribution of the Kmr gene to 1.2 plasmid stability, pBLU-KP and pBLU-KS were constructed and introduced into yeast cells, where they 1 resolved into pBLUR-KP and pBLUR-KS. The Kmr gene orientation had no impact on their stability in glucose e

r 0.8 u

t medium, since the plasmid loss rates of these vectors l u c

f 0.6 were identical. When cultivated for 36 generations on o

l galactose, plasmid loss rates were 0.07% for pBLUR-KP m /

U 0.4 and 0.93% for pBLUR-KS, suggesting that prolonged generation time does not contribute to plasmid instability 0.2 either. It should be pointed out that plasmid pBLUR-KP exhibited considerable discrepancy of stability while cul- 0 tivated in galactose medium, both within a single experi- pGoBR-2KS pGoBR-2 ment and between independent experiments. Plasmid loss rates are given in Table 3.

b In pGoB-2KS and pGIFN constructs, the KanMX4 cassette was cloned together with the inverted TRP5 terminator sequence from transient vector YcpKan (Todorović et al., 2000). Inverted TRP5 was 2 ter regarded as not important for heterologous gene expres- 1.8 1.6 sion. The pBLU-KTP and pBLU-KTS constructs were

8 created in order to test the possible influence of inverted - 1.4 E 0 . TRP5ter and the KanMX4 cassette on plasmid stability, 1 1.2

l since such an assembly of genetic elements exists in the l 1 e c / 0.8 most unstable pGoB-based vectors. The stability of U 0.6 pBLUR-KTP/S, both on glucose and galactose, did not r 0.4 differ from that of the other Km constructs. 0.2 Efficient termination of foreign gene transcription is 0 one of the factors that generally influence gene expres- pGoBR-2KS pGoBR-2 sion in yeast (Bijvoetet al., 1991). Because the BLITZ cassette contains a promotor and a terminator of different strengths, we tested the possibility that ineffi- Fig. 3 - Comparison of PGA activities between clones carrying pGoBR- cient termination could contribute to plasmid instability 2KS and pGoBR-2. 3a - Overall production of rPGA in CBL1- 30/pGoBR-2KS and CBL1-30/pGoBR-2 clone 72 h after the induction. by carrying on transcription through the STB locus. The 3b - Production of rPGA per PGA+ cell. GAL1-10UAS sequence is one of the strongest yeast acti- 8 N. STANKOVIĆ ETAL.

Table 3. Loss rates of pBLU-based model vectors expressed as % of cal and stable PCN values which were considerably plasmid free cells in population after one division. reduced once selective pressure had been removed. In PLR - plasmid loss rate, calculated as described in Materials and Methods. pGoB-based plasmids, PCN was irregular and varied (Fig. 4). Cells cultivated in glucose had a lower, yet more plasmid media PLR/generation (%) stable PCN. The pGIFNR plasmid appeared to be present in a considerably lower copy number than the isogenic pGoBR-2KS. YPD 0 DISCUSSION pBLUR-D YPGal 0.07 Production of heterologous proteins in baker’s yeast YPD 0.53 ± 0.05 is influenced by many factors (for review see pBLUR-KP YPGal 0.07 ± 0.32 Romanoset al., 1992). Gene dosage is considered to be one of the most important. When expression is from YPD 0.53 ± 0.05 YEp vectors, gene dosage depends on plasmid stability in the population and on plasmid copy number. pBLUR-KS YPGal 0.93 ± 0.03 Cultivation on a neutral carbon source in the pres- YPD 0.32 ± 0.05 ence of G418 showed that the population of pBLU-based pBLUR-TK YPGal 0.36 ± 0.17 clones and pGIFNR contained 1-10% of plasmid-free segregants. Monitoring of plasmids with auxotrophic YPD 0.66 ± 0.13 selective markers (Gietz & Sugino, 1988; Christianson et al., 1992) revealed that even under pBLUR-KT5P YPGal 0.64 ± 0.14 selection, plasmid-free cells encompassed 5-30% of the population. This represented the balance between gener- YPD 0.61 ± 0.05 ation of new and loss of old plasmid-free segregants. pBLUR-KT5S YPGal 0.72 ± 0.67 However, the pGoBR-2KS clone contained 40% of plasmid free segregants after 10 generations. Plasmids that are pGoB-based usually have high average copy numbers vating sequences, but the TRP5 terminator corresponds to in the cell, but clonal variation may lead to differences in a TRP5 promoter of medium strength. We replaced stability between clones of the same construct. TEF from A. gossypii in the KanMX4 cassette Presumably, those clones with low PCN lose plasmids ter rapidly, since plasmids that have lowered PCN also tend (Stainer& Philippsen, 1994) with TRP1 from ter to show segregational instability (Futcher & Cox, S. cerevisiae to mimic conditions of intensive transcrip- 1984; and Kirkpekar & Gulløv, 1996). In the tion of a foreign gene with a promoter and terminator of case of pGoBR-2KS, clonal variation seems to be signif- different origin and strength. The plasmid pBLU-TK was icant. constructed and its stability tested. Plasmid loss rates were the same as in constructs carrying the Kmr gene In culture phase-induction experiments (Ljubi- jankić, unpublished results), the CBL1-30/pGoBR-2 with TEFter. culture induced by galactose in early growth stages con- Copy number tained a smaller number of plasmid-bearing cells. Most of the plasmid-bearing cells as well as the highest yield The plasmid copy number (PCN) changed in the of recombinant PGA coincided with the minimal number population during nonselective growth, as did the per- of divisions that the culture underwent during the induc- centage of plasmid-carrying cells. Since the number of tion. The considerable difference in stability of pGoB plasmid-carrying cells declined after every doubling, plasmids cultivated on glucose vs. ones cultivated on thereby reducing the number of cells able to segregate in galactose is not uncommon, since other 2μm-based future divisions, PCN was normalized for plasmid loss expression vectors, as well as ARS vectors containing a rates (Table 4). All pBLU-based plasmids showed identi- galactose-dependent promoter, show reduced stability, EFFECT OF KANAMYCINE RESISTANCE MARKER 9 depending on the carbon source (Bitter & Egan, gene in the population at the time of induction, since sim- 1988; Kirkpekar & Gulløv, 1996). ilar rPGA production per cell suggests an equal ability of the single clone to produce recombinant protein. Plasmid loss in CBL1-30/pGoBR-2KS and CBL1- 30/pGIFNR clones during growth in glucose could be When recombinant protein is produced from a vec- additionally attributed to constitutive expression of Kmr tor containing the BLITZ cassette, such as pGoB-based and overabundance of its product. The KanMX4 cassette vectors, plasmid instability is also connected to the actu- is effectively expressed owing to its strong TEF promot- al process of foreign gene expression. The cassette con- er. A single copy of this cassette conferred resistance in tains 365 bp of the intergenic region, GAL1-GAL10 with A. gossypii to geneticin up to 8 mg/ml (Steiner & a UAS element, the TATA box and leader sequence of Philippsen, 1994). Efficiency of the TEF promoter MFα1, and the terminator region of the yeast TRP5 gene in baker’s yeast is enough to allow resistance to 200 (Ludwig et al., 1993). Neither of these genetic ele- µg/ml G418, even in the presence of a single copy [(as ments influences plasmid stability simply by its presence shown for the KanMX4 cassette used in ORF deletions [GAL1-10UAS was cloned in different orientation on plas-

Table 4. Estimation of plasmid copy number (PCN). pBLU - pBLU-based plasmids; I - PCN after 12 generations of growth; II - PCN after 24 generations of growth; III - PCN after 36 generations of growth. Note:Where two measurements were made, both values are listed, and the average value is shown in parentheses.

Medium YPRafG418 YPD YPGal

batch I I II III I II III

pBLU 50 15-20 15-20 15-20 15-20 15-20 15-20

27 24 12 21 1 1 3

pGIFNR 4 3 3 1 10 3 4

(15) (14) (7) (11) (6) (2) (4)

pGoBR-2KS 42 31 29 59 220 17 20

and construction of ORF replacement cassettes in Saecharomyces cerevisiae, Wach et al., (1996)]. mids pBLU-G and pBLU-GC (not shown), TRP5ter on

Therefore, cells could afford to lose most of their plasmid plasmids pBLU-KTP/S]. The GAL1-10UAS sequence is molecules and still remain G418-resistant. A considerable one of the strongest yeast-activating sequences, but the effect is also realised through accumulation of the Kmr TRP5 terminator corresponds to a TRP5 promoter of gene product, APT, which remains in the cell after loss of medium strength. It was previously shown that a bidirec- the plasmid. Aminoglycoside phosphotransferase is sta- tional promoter of medium capacity, TRP1ter, was not ble in yeast cells, and its half-life was calculated to be sufficiently effective in termination of transcription from about 7.5 h (Hadfield et al. 1990). Thus, cultures at the GAL1 gene (Irniger et al., 1991). We did not dis- the optimal growth rate should pass another two or three cern that discordance in strength and origin of the pro- divisions before the steady-state level of residual APT is moter and terminator played any role in plasmid instabil- reduced by one half. ity (to judge from model plasmid pBLUR-TK). This sug- The low activity of the pGoBR-2KS clone in pro- gests that, in the case of pGoBR-2KS and pGIFNR, the ducing PGA is probably due to diminished stability on intensity of expression from foreign sequences is the glucose and the small number of cells containing the pac main reason for plasmid instability. The transcriptional status and nature of both heterologous genes (gene of 10 N. STANKOVIĆ ETAL.

interest and Kmr marker) probably impaired the mainte- nance of expression vectors, although all vital sequences of 2µm remained intact. In a comparative study of four auxotrophic markers, Ugolini et al.(2002) demon- strated how the level of expression on a plasmid is inversely correlated with plasmid stability and copy number, and - hypothetically - in accordance with its strong secondary, hairpin like, DNA structure. The synergistic

Fig. 4 – Southern blot analysis of total DNA from Saecharomyces cerevisiae clones carrying plasmids pBLUR-KP, pGIFNR, and pGoBR- 2KS. Each lane contains a signal from the plasmid DNA and a signal from the chromosomal fragment with a single-copy VMA22 gene. Lane

1: total yeast DNA isolated from the YPRafG418 culture; lanes 2-4: total yeast DNA isolated from the YPD culture (after 12, 24, and 36 generations of growth, respectively); lanes 5-7: total yeast DNA isolated from the YPGal culture (after 12, 24, and 36 generations of growth, respectively). * - in lane 5, the signal from VMA22 is partly masked by incom- pletely digested pGoBR-2KS. effect of strong expression from GAL1-10 and TEF UAS level of resistance proved to be an inadequate tool for promoters could impair replication and partition func- monitoring baker’s yeast clones carrying multicopy vec- tions by disturbing plasmid secondary structure. Size of tors for heterologous protein production. This should be the pac gene apparently plays a role in the high instabili- taken into account as a general principle in the choice of ty of pGoBR-2KS, as it is the feature most divergent from S. cerevisiae selective markers. Ideally, resistance should its more stable twin plasmid, pGIFNR. be provided not by a high level of expression, but also The stability of pBLU-based plasmids was reduced through a large number of gene copies, as in the case of =1% by expression of the gene for kanamycin resistance. the auxotrophic markers leu-2d and ura3-d (Beggs, r 1978; Loison et al., 1989). In vectors for rPGAP. rett. expression, the effect of Km introduction was manifested as a decrease in the yield of Abbreviations:Ap – ampicillin; APT - aminoglycoside enzyme, since recombinant product formation mirrored phosphotransferase; Flp - S. cerevisiae recombinase; FRT - Flp vector stability. The KanMX4 cassette conferring a high recognition target; Km - kanamycin; MFα1 - mating factor α; EFFECT OF KANAMYCINE RESISTANCE MARKER 11

pac – gene for penicillin G amidase; PGA – penicillin G ami- Hanahan, D. (1983). Studies on transformation of Escherichia coli with dase; STB - 2μ stabilization locus; TEF - translation elongation plasmids. J. Mol. Biol. 166, 557-580. factor 1 alpha; UAS - upstream activating sequence; VMA22 - Inoue, H., Nojima, H. and Okayama, H. (1990). High efficiency trans- gene for one of the subunits of yeast V-ATPase. formation of Escherichia coli with plasmids. Gene 96, 23-28.

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УТИЦАJ МАРКЕР ГЕНА ЗA КАНАМИЦИНСКУ РЕЗИСТЕНЦИJУ НА СТАБИЛНОСТ 2m EКСПРЕСИОНИХ ПЛАЗМИДА

НАДА СТАНКОВИЋ, БРАНКА ВАСИЉЕВИЋ и †ГОРАН ЉУБИЈАНКИЋ

Институт за молекуларну генетику и генетичко инжењерство, 11010 Београд, Србија

У овом раду описан је утицај гена за канамицинску рација. Примена селективног притиска на сојеве који резистенцију (Kmr) на одржавање 2mm плазмида у производе рекомбинантну пеницилин G амидазу (rP- Saccharomyces cerevisiae. Присуство овог маркер гена GA) није довела до очекиваног повећања приноса доводи до губитка стабилног модел-вектора кон- протеина. Испитивањем утицаја самих генетичких стантном стопом независном од извора угљеника и елемената за производњу хетерологних протеина на стопе раста културе. Код сојева за синтезу хетеролог- стабилност вектора показано је да најјачe дестабили- них протеина са галактозног промотора (GALUAS) увођење Kmr резултира „чишћeњем“ од квашчевих шућe деjство имa присуство и eкспреcиjа страног ге- епизомалних плазмида (YEp) у свега неколико гене- на.